This page contains proprietary information. Please do not distribute for any purposes other than evaluation and review. PROJECT SUMMARY/ABSTRACT Lung cancer is the most common primary cancer to spread to the brain. The median survival for patients with non-small cell lung cancer (NSCLC) brain metastases is about 4 months. While radiotherapy remains a first- line treatment for patients with multiple brain metastases, recurrence is observed in over 40% of patients. A new therapeutic agent is desperately needed in order to find and eradicate these remaining brain micrometastases post-radiotherapy. To address this need, we propose using neural stem cells (NSCs) as a targeted drug delivery system to scavenge for remaining lung cancer micrometastases in the brain after radiotherapy. NSCs have demonstrated a remarkable, innate ability to selectively migrate to tumors. When programmed to produce cytotoxic proteins, NSCs have been proven to migrate to and kill glioblastoma tumors. Despite the promise of NSCs for tumor-targeted treatment, NSCs have proven difficult to harvest. Allogeneic cells activate the immune response, promote clearance of NSCs, and decrease their therapeutic window. Autologous NSCs are needed to avoid this immune response but are impractical to collect due to location and quantities found in the brain. In order to combat this problem, we have previously developed a method of rapidly transdifferentiating human fibroblasts into induced neural stem cells (hiNSCs) using a lentivirus containing the gene to induce expression of the multipotency transcription factor, Sox2. The hiNSCs are a personalized, tumor-homing therapeutic cell line. However, previous research surrounding hiNSCs has focused on the treatment of primary brain cancer. This work will be the first to investigate hiNSCs as a method of eradicating far-reaching NSCLC micrometastases found in the brain. Moreover, we will combine this innovative therapy with well-established radiotherapy regimens in order to develop a model that reflects current treatment regimens. In order to assess the potential of therapeutic hiNSCs as an adjuvant treatment for NSCLC brain micrometastases, we will perform treatment optimization in vitro, use in vivo studies to determine the migration, persistence, and efficacy of hiNSC following radiotherapy, and single-cell mRNA sequencing to elucidate tumor and tumor bed heterogeneity, particularly regarding sensitization or resistance, that occurs after this combination therapy. If successful, this will be important foundational work in the development of a new, much-needed therapeutic agent to scavenge for NSCLC micrometastases remaining after radiotherapy, thus reducing the risk of tumor recurrence and decreasing the mortality of NSCLC.